HSP70 inhibitors upregulate prostaglandin E1-induced synthesis of interleukin-6 in osteoblasts

Interleukin-6 (IL-6) is a pro-inflammatory and bone-resorptive cytokine that also regulates bone formation. We previously showed that prostaglandin E1 (PGE1) induces the synthesis of IL-6 by activating p44/p42 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAPK in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether heat shock protein 70 (HSP70), a molecular chaperone that coordinates protein folding and homeostasis, affects PGE1-stimulated IL-6 synthesis in MC3T3-E1 cells through the MAPK activation. The osteoblast-like MC3T3-E1 cells were treated with HSP70 inhibitors—VER-155008 and YM-08—, PD98059, SB203580 or SP600125 and then stimulated with PGE1. IL-6 synthesis was evaluated using an IL-6 enzyme-linked immunosorbent assay kit. IL-6 mRNA expression was measured by real-time RT-PCR. The phosphorylation of p38 MAPK was evaluated by Western blotting. We found that VER-155008, an HSP70 inhibitor, enhanced the PGE1-stimulated IL-6 release and IL-6 mRNA expression. YM-08, another HSP70 inhibitor, also enhanced PGE1-stimulated IL-6 release. PD98059, a p44/p42 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, upregulated PGE1-stimulated IL-6 release. On the other hand, SB203580, a p38 MAPK inhibitor, suppressed PGE1-stimulated IL-6 release. YM-08 stimulated the PGE1-induced phosphorylation of p38 MAPK. SB203580 suppressed the amplification by YM-08 of the PGE1-stimulated IL-6 release. Our results suggest that HSP70 inhibitors upregulate the PGE1-stimulated IL-6 synthesis through p38 MAPK in osteoblasts and therefore affect bone remodeling.


Introduction
Biological stresses such as heat, hypoxia, and extracellular stresses prompt cells to produce heat shock proteins (HSPs), whose cytoprotective functions protect unfolded proteins from aggregating [1]. These molecular chaperones are divided into six groups based on their molecular weight: HSP27, HSP40, HSP60, HSP70, HSP90, and HSP110 [2]. Among them, HSP70 is constitutively expressed in unstressed cells and acts as an ATP-dependent molecular chaperone [3]. HSP70 also contributes to the translocation of the synthesized proteins into organelles across their membranes [4]. HSP70 has been implicated in various diseases including neurodegenerative disease and Alzheimer's disease [5,6]; it may also become a therapeutic target of cancer because HSP70 expression is elevated in tissue from the patients with hepatocellular carcinoma and breast cancer [7,8].
Both osteoblasts and osteoclasts tightly coordinate bone metabolism by forming and resorbing bone, respectively [9]. In the adult skeletal system, old bone is continuously regenerated into new bone by resorption in a process called bone remodeling that maintains the volume and the strength of bone [10]. Disordered bone remodeling, however, causes metabolic bone diseases such as fracture healing distress and osteoporosis. Key members in this process include cytokines, hormones, and growth factors play important roles in bone remodeling [11,12].
Specifically, interleukin-6 (IL-6) is a multifunctional cytokine that promotes B cell differentiation and T cell activation and induces acute phase proteins [13]. IL-6 not only promotes osteoclastogoenesis and bone resorption indirectly by stimulating receptor activator of nuclear factor κB ligand (RANKL) expression by osteoblasts but also influences bone formation as an osteotropic factor under conditions of increased bone turnover, such as fracture healing [14,15]. The soluble IL-6 receptor binds IL-6 to promote osteoblast differentiation via the gp130 receptor [16].
In the present study, we investigated how HSP70 inhibitors affect the release of IL-6 in PGE 1 -induced osteoblast-like MC3T3-E1 cells. We found that HSP70 inhibitors upregulated the PGE 1 -stimulated IL-6 synthesis through p38 MAPK in osteoblasts.

Cell culture
Cloned osteoblast-like MC3T3-E1 cells were established from neonatal mouse calvaria and maintained as previously described [25]. Briefly, the cells were cultured in 10% fetal bovine serum (FBS)-containing α-minimum essential medium (α-MEM) at 37˚C in humidified atmospheric air with 5% CO 2 . The cultured cells were seeded on 90-mm diameter dishes (2 × 10 5 cells/dish) in α-MEM containing 10% FBS. The medium was replaced with α-MEM containing 0.3% FBS at 5 days post-seeding. These cells were used for experiments after 48 h.

Assay for IL-6
The cultured MC3T3-E1 cells were pretreated with various doses of VER-155008, 10 μM of YM-08, 50 μM of PD98059, 30 μM of SB203580, and 10 μM of SP600125 for 60 min [23,24], and then stimulated with 10 μM of PGE 1 in 1 ml of α-MEM containing 0.3% FBS for the indicated periods. The conditioned medium was collected at the end of incubation, and the IL-6 concentration was measured using a mouse IL-6 ELISA kit according to the manufacturer's instructions [19].

Real-time RT-PCR
The cultured MC3T3-E1 cells were pretreated with 30 μM of VER-155008 or vehicle for 60 min and then stimulated with 10 μM of PGE 1 or vehicle in α-MEM containing 0.3% FBS for 2 h [19,23]. Trizol Reagent (Invitrogen; Thermo Fisher Scientific, Inc. Heysham, Lancashire, UK) and Omniscript Reverse Transcriptase kit (Qiagen Inc., Valencia, CA, USA) were respectively used to isolate total RNA and transcribe it into complementary DNA. Real-time RT-PCR was performed using a LightCycler system (version 3.5; Roche Diagnostics, Basel, Switzerland) in capillaries and Fast Start DNA Master SYBR Green I provided with the kit (Roche Diagnostics). Sense and antisense primers for mouse IL-6 mRNA (primer set ID: MA039013) and GAPDH mRNA (primer set ID: RA015380) were purchased from Takara Bio Inc. (Tokyo, Japan). The amplified products were determined using a melting curve analysis. The IL-6 mRNA level of IL-6 was normalized to that of GAPDH [24]. We have already reported using ELISA that 30 μM of VER-155008 significantly amplified the TGF-β-stimulated VEGF release in osteoblast-like MC3T3-E1 cells [23]. We also demonstrated using real-time RT-PCR that 10 μM of VER-155008 increased the TGF-β-stimulated VEGF mRNA expression levels in the same cells [23]. Therefore, we adopted the same VER-155008 concentrations in this study as described in the aforementioned study for the real-time RT-PCR experiments.

Western blot analysis
The cultured cells were pretreated with various doses of YM-08 for 60 min and then stimulated with 10 μM of PGE 1 or vehicle in 1 ml of α-MEM containing 0.3% FBS for 20 min. The cells were then lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM Tris/HCl, pH 6.8, 2% SDS, 50 mM dithiothreitol, and 10% glycerol. SDS-polyacrylamide gel electrophoresis (PAGE) was performed using the Laemmli method in 10% polyacrylamide gels [26]. The protein was fractionated and transferred onto an Immun-Blot polyvinylidine difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% fatfree dry milk in Tris-buffered saline-Tween (TBS-T; 20 mM Tris/HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 1 h before incubation with primary antibodies. A Western blot analysis was performed as described previously [27] using antibodies against phospho-specific p38 MAPK, p38 MAPK, and actin as primary antibodies with peroxidase-labeled antibodies raised in goat against rabbit IgG (KPL, Inc., Gaitherburg, MD, USA) being used as secondary antibodies. The primary and secondary antibodies were diluted in TBS-T with 5% fat-free dry milk to optimal concentrations. An X-ray film using an electrochemiluminescence Western blotting detection system was used to visualize peroxidase activity on the membrane; each protein was detected on different gels.

Densitometric analysis
A densitometric analysis of the Western blots was performed with a scanner and image analysis software program (ImageJ version 1.49, NIH, Bethesda, MD, USA). The phosphorylated levels were calculated as follows: the background-subtracted signal intensity of each phosphorylation signal was normalized to the respective intensity of total protein and plotted as the fold increase relative to that of the control cells without stimulation.

Statistical analysis
The data were analyzed by an analysis of variance followed by Bonferroni method for multiple comparisons between pairs, and p < 0.05 indicated statistical significant. All data are presented as the mean ± standard error of the mean (SEM) of triplicate determinations from three independent cell preparations.

Effect of VER-155008 on the PGE 1 -stimulated IL-6 release in MC3T3-E1 cells
We investigated how VER-155008, an inhibitor of HSP70 [28], affected the PGE 1 -stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We found that VER-155008 significantly enhanced PGE 1 -stimulated IL-6 release (Fig 1A). This amplification was time-dependent up to 36 h, which showed a 500-fold increase in the PGE 1 -stimulated effect. In addition, considerable statistical significance was observed between the group of 30 μM VER-155008 alone (□) and the control group (�) in the IL-6 release at 24 h (Fig 1A). We also found that this enhancement of PGE 1 -stimulated IL-6 release was dose-dependent between 1 and 30 μM in these cells ( Fig 1B); VER-155008 at 30 μM elicited an approximately 300-fold increase in the PGE 1 -stimulated IL-6 release.

Effect of VER-155008 on the PGE 1 -induced expression levels of IL-6 mRNA in MC3T3-E1 cells
We next quantified PGE 1 -induced mRNA expression of IL-6 with RT-PCR to determine if transcription mediated VER-155008's amplification of PGE 1 -stimulated IL-6 release. VER-155008 enhanced mRNA expression levels of PGE 1 -induced IL-6 155008 (Fig 2). Interestingly, VER-155008 alone stimulated IL-6 mRNA expression levels although VER-155008 alone did not affect the IL-6 release.

Effects of YM-08 on the PGE 1 -stimulated phosphorylation of p38 MAPK in MC3T3-E1 cells
After finding that only SB203580 weakened PGE 1 -induced IL-6 synthesis, we further examined the effect of YM-08 on the PGE 1 -induced phosphorylation of p38 MAPK. Concentrations of YM-08 between 10 and 70 μM significantly enhanced the PGE 1 -induced phosphorylation of p38 MAPK (Fig 4).

PLOS ONE
Effects of HSP70 inhibitors on interleukin-6 synthesis in osteoblasts of IL-6 mRNA, which VER-155008 further enhanced. Our findings suggest that HSP70 inhibitor enhances PGE 1 -stimulated IL-6 release at a point upstream of transcriptional levels in osteoblast-like MC3T3-E1 cells. However, we observed considerable statistical significance between the group of 30 μM VER-155008 alone (□) and the control (�) in the case of IL-6 release at 24 h, which seemed to be consistent in part with the VER-155008 effect on the IL-6 mRNA expression. Regarding the IL-6 mRNA expression levels, VER-155008-mediated upregulation by itself was higher than that influenced by PGE 1 alone, whereas the PGE 1 -mediated IL-6 release levels were higher than those affected by VER-155008. Concerning the VER-155008 effect on mRNA expression, VER-155008 reportedly enhances toll-like receptor 5 (TLR5) mRNA expression but reduces TLR5 cell surface expression in human myeloid leukemia THP-1 cells, suggesting that suppressing the HSP70 inhibitor-related chaperone function could prevent mature TLR5 traffic from the endoplasmic reticulum (ER) to the cell surface [33]. Therefore, the discrepancy between how VER-155008 and PGE 1 affect the IL-6 mRNA expression levels and IL-6 release might be caused by the VER-155008-induced suppression of mature IL-6 translocation from the ER to secretory granules by VER-155008 in osteoblasts.
We sought to elucidate the exact mechanism by which HSP70 inhibitor upregulates PGE 1stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We found that YM-08 markedly enhanced the PGE 1 -induced phosphorylation of p38 MAPK. SB203580 also suppressed YM-08's amplification of PGE 1 -stimulated IL-6 release. Based on these findings, it is likely that the activation of p38 MAPK in osteoblast-like MC3T3-E1 cells mediates this process. Related to the PGE 1 -elicited p38 MAPK pathway in osteoblast-like MC3T3-E1 cells, we have previously reported that cAMP/protein kinase A pathway exists upstream of p38 MAPK and is involved in p38 MAPK activation [34]. The adenylate cyclase activity triggers cAMP formation from ATP, resulting in protein kinase A activation. In this study, HSP70 inhibitor increased the PGE 1 -induced p38 MAPK phosphorylation and upregulated the PGE 1 -stimulated IL-6 synthesis. Taking these findings into account as a whole, it is likely that HSP70 might affect cAMP/ protein kinase A pathway and consequently regulate p38 MAPK phosphorylation in osteoblast-like MC3T3-E1 cells. Further investigation would be required to elucidate the possible underlying mechanism of how HSP70 regulates p38 MAPK phosphorylation in osteoblasts. However, only YM-08 but not VER-155008 was used to evaluate PGE 1 -stimulated p38 MAPK phosphorylation in this study. Unfortunately, no data on the use of VER-155008 to confirm the enhancing effects of YM-08 on the PGE 1 -stimulated p38 MAPK phosphorylation is available. Further investigations using VER-155008 for the PGE 1 -stimulated p38 MAPK phosphorylation would be necessary to provide additional support to our findings. IL-6 is a pro-inflammatory cytokine that governs bone remodeling under physiological and pathological conditions by inducing RANKL expression and therefore osteoclastogenesis as well as promoting bone resorption [14,15]. A recent study suggests that IL-6 is necessary for bone formation and functions as an osteotropic factor during increased bone turnover [15]. On the other hand, prostaglandin, a bone-resorptive cytokine, also mediates bone remodeling and formation [35]. Bone resorption by osteoclasts initiates bone remodeling, after which osteoblasts form bone. Proper bone remodeling is essential for maintaining bone quality and bone volume. IL-6 is currently known to mediate bone remodeling from the viewpoint of whole bone metabolism, and osteoblast-like MC3T3-E1 cells normally express HSP70 even when not stimulated [36]. Accounting for these findings, our results show that HSP70 functions in not only protein folding and proteostasis but also bone metabolism. HSP70 is implicated in neurodegenerative diseases and cancer; HSP70 activity is a potential drug target [5]. We showed that HSP70 inhibitors enhance PGE 1 -stimulated IL-6 synthesis in osteoblasts and therefore could affect bone metabolism as a modulator of bone remodeling through IL-6 synthesis in osteoblasts.
In conclusion, our results strongly suggest that the HSP70 inhibitors upregulate PGE 1 -stimulated IL-6 synthesis through p38 MAPK in osteoblasts. Further investigations should further explore how HSP70 functions in osteoblasts.